The methylation of histone lysine residues has been correlated to numerous phenotypes of cancer. Histone lysine residues can have up to three methyls added and each state can have clearly different cellular roles. For years, researchers in epigenetics (including those focused on epigenetic processes in cancer) have used techniques that rely on antibodies or radioactivity to measure the methylation state of a given lysine; however, these are neither comprehensive nor quantitative. What has become an alternative approach to get around these limitations is the direct use of mass spectrometry, but the problem with this approach is that differentially methylated peptides do not ionize the same and CANNOT be directly compared as a measurement of activity. Therefore, the field of cancer epigenetic research and epigenetics as a whole needed a comprehensive method to simultaneously monitor demethylase/methyltransferase reaction intermediates (i.e., different methyl states on a lysine) in a quantitative manner. In 2011, we provided a novel approach for the comprehensive and quantitative measurement of lysine methylation states, which is called MassSQUIRM (Mass Spectrometric Quantitation Using Isotopic Reductive Methylation). MassSQUIRM utilizes the chemical incorporation of isotopically heavy methyl groups on lysines to convert all reaction intermediates (un- and monomethyl) to fully dimethyl lysines (differing only by hydrogen and deuterium - which does not affect ionization properties in mass spectrometry). A comparison of peptide intensities of the mixture of heavy and light species allows for comprehensive (un-, mono- and dimethyl states) quantitation of lysine methylation. We recently published the MassSQUIRM technique, and in this application we outline how we will evaluate it in a cancer relevant context in order to ultimately develop a kit for cancer research. Our overall goal is to provide a MassSQUIRM kit to cancer researchers to assay demethylation and methylation (un-, mono- and dimethylation specifically) of lysine residues in proteins correlated to particular cancer phenotypes. To validate the MassSQUIRM approach for its use in cancer research, we will pursue the following Aims: Aim 1. Determine the general applicability of MassSQUIRM by assaying a panel of histone lysine demethylases and methyltransferases. Aim 2. Evaluate the effectiveness of using MassSQUIRM to assay LSD1 activity from cell lysates. Aim 3. Optimize MassSQUIRM for lysine demethylation screening with a panel of LSD1 inhibitors.